1. Field of the Invention
This invention relates to use of DNA fragments having promoter activity from basidiomycetes, particularly Coriolus hirsutus and Lentinus edodes in production of useful polypeptides such as lignin degrading enzymes. More specifically, this invention relates to a Coriolus hirsutus host cell transformed with a vector including a Coriolus hirsutus-derived ras promoter region or a Lentinus edodes-derived ras or priA promoter region, and to a method for producing useful polypeptides using the host cell.
According to this invention, polypeptides such as lignin degrading enzymes, the production of which has been considered to be difficult, can be supplied stably and in large quantity by genetic recombination technique using the host-vector system, particularly basidiomycete-derived host-vector system, of the present invention.
2. Background Art
Conventionally, wood pulps have been produced popularly by methods of chemically treating woods. However, from the viewpoint of environmental problems or the like, there has been an attempt to produce a cellulose pulp by inoculating a white rotting fungus into a lignocellulose substance of wood or the like then culturing the fungus to degrade lignin (Japanese Patent Application Laid-open No. 46,903/1975). However, the white rotting fungus used in this method has problems that the coexisting carbohydrates are degraded or, in cases of using cellulase-deficient mutants, their native lignin degrading ability becomes weaken, so that this method has not yet been put into practice.
In order to solve such problems as above, there has been an attempt to make a lignin degrading enzyme from a white rotting fungus act on a lignocellulose substance to selectively degrade lignin alone (Science, 221, 661 (1983)). This report, though mainly a lignin model compound is used as a substrate, is the first one in the world in which a lignin degrading enzyme was isolated and purified. This enzyme is an extracellular enzyme which Phanerochaete chrysosporium produces, and the main characteristics of the enzyme are as follows: the optimum pH is 3.0; it is an iron-containing enzyme; the molecular weight is 41,000 to 42,000 Da; hydrogen peroxide is necessary for enzymatic reaction; and it is confirmed to act on a compound formed by replacing the phenolic hydroxyl group at position 4 of a lignin model compound with a methoxyl group. This enzyme is lignin peroxidase, and the existence of plural isozymes are known (FEBS Lett., 169, 247 (1984)). Lignin peroxidase is found from many wood-rotting fungi such as Coriolus versicolor, Bjerkandera adusta, etc. other than the above, some of which have been purified.
On the other hand, lignin degrading enzymes to be produced by Coriolus hirsutus and Lenzites betulina are also known (Japanese Patent Application Laid-open Nos. 220,190/1987 and 220,189/1987), which are phenol oxidases, and their main characteristics are that the optimum pH is 4.5, that they are copper-containing enzymes, that the molecular weight is approximately 63,000 Da or approximately 65,000 Da, that the isoelectric point is around 3.5, that oxygen is necessary for the enzymatic action, and that they do not act on a compound formed by replacing the phenolic hydroxyl group at position 4 of a lignin model compound with a methoxyl group but on a phenolic lignin model.
Besides, manganese peroxidase is one of typical lignin degrading enzymes, the main characteristics of which are that the molecular weight is 46,000 Da or less, that it is presumed to be an iron-containing enzyme, that hydrogen peroxide is necessary for the enzymatic action, that the enzyme reaction is Mn(II)-dependent, that it is confirmed to never act on a compound formed by replacing the phenolic hydroxyl group at position 4 of a lignin model compound with a methoxyl group but on a phenolic lignin model, and therefore it is an enzyme having properties quite different from those of the lignin peroxidase.
Up to now, the production of varieties of polypeptides by recombinant DNA techniques has been carried out using a host system centering around Escherichia coli (also designated E. coli). However, there are many cases where E. coli is not appropriate as a host. For example, there are many problems, in cases of producing useful polypeptides (e.g., enzymes) from a higher animal such as human being or the like, that the polypeptide of interest is not produced as an active protein, and that a large number of toxic substances other than the polypeptide of interest are produced making the purification of the target product very difficult. As alternative means for solving these problems, production methods using yeast, a lower eukaryote, as a host have been studied extensively, which however newly bring about a problem of low productivity. For the purpose of polypeptide production, transformation systems using higher eukaryotes such as filamentaous fungi (e.g. Aspergillus) and basidiomycetes (e.g. Phanerochaete and Coriolus) as hosts have also been developed, and the production of lignin degrading enzymes using the system has been studied.
Basidiomycetes belong to eukaryotes and are considered more closely related to animal cells than yeast (T. L. Smith, Proc. Natl. Acad. Sci. USA, 86, 7063 (1989)). Coriolus hirsutus having a strong lignin degrading ability is a basidiomycete belonging to the genus Coriolus, whose host-vector system has been developed by the present inventors employing recombinant DNA techniques. As a result, the present inventors succeeded in the production of lignin peroxidase that had been considered to be difficult so far (Japanese Patent Application Laid-open No. 054,691/1994). However, the promoter region used therein was a promoter region of the ornithine carbamoyltransferase gene (hereinafter referred to as xe2x80x9cOCT genexe2x80x9d), which is a gene for amino acid synthetase, or a promoter region of the phenol oxidase gene participating in lignin degradation, so there were problems that the productivity of the lignin peroxide was as low as that produced by a wild strain IFO4917 cultured in a lignin peroxidase production medium (low carbon and nitrogen sources), and that it took a lot of time for gene expression because the target enzyme was obtained as a secondary metabolite. In addition, it has been reported that a promoter, which is provided for enzyme protein production systems by genetic recombination of other organism species (e.g. filamentous fungi), was not function in basidiomycetes (A. Lorna et. al., Curr. Genet., 16, 35 (1989)). Furthermore, ArgB gene from Aspergillus nidulans did not function in Coriolus hirsutus (A. Tsukamoto et al., U.S. Pat. No. 5,362,640).
Therefore, the present inventors obtained a promoter for constitutively expressing Coriolus hirsutus glyceraldehyde-3-phosphate dehydrogenase (GPD) gene, ligated thereto a structural gene with signal peptide coding sequence of a high temperature-induced lignin peroxidase gene (Japanese Patent Application Laid-open No. 260,978/1993) or a manganese per-oxidase gene (Japanese Patent Application Laid-open No. 308,581/1996) cloned from the Coriolus hirsutus, transformed the ligated product into an ornithine carbamoyltransferase-deficient Coriolus hirsutus mutant, and thereby succeeding in obtaining a strain capable of highly producing lignin peroxidase or manganese peroxidase (Japanese Patent Application Laid-open No. 47,289/1997).
However, in spite of the above technical proposal, a growing interest is taken in a promoter having a strong transcription activity and enabling high expression of a useful polypeptide.
Under such circumstances, the present inventors noticed a basidiomycete host-vector system and searched for various promoters functioning in this system.
Therefore, an object of the present invention is to provide a promoter for allowing a host such as basidiomycete or to produce a useful polypeptide in a large quantity.
Another object of the present invention is to provide a host-vector system including the promoter and a hyperexpression and production method of a useful polypeptide utilizing the system.
The present inventors noticed a constitutively expressing Coriolus hirsutus ras gene and Lentinus edodes ras and priA genes, the priA gene being expresses highly when a fruit body primoridium is formed in Lentinus edodes, cloned a DNA fragment encoding the ras gene or the priA gene from a Coriolus hirsutus or Lentinus edodes chromosomal DNA restriction fraction, sequenced a promoter region upstream of this gene, and found for the first time that this promoter region was effective for the expression of a gene encoding a useful polypeptide in a hostvector system, particularly Coriolus hirsutus host-vector system. Furthermore, the present inventors ligated a structural gene with signal peptide coding sequence of a manganese peroxidase gene, high temperature-induced lignin peroxidase gene (Japanese Patent Application Laid-open No. 260,978/1993), or laccase gene, the structural gene having been cloned from Coriolus hirsutus, to a site downstream of the above promoter region, introduced the same into an OCT-deficient Coriolus hirsutus mutant, and thereby succeeded in obtaining a manganese peroxidase-, lignin peroxidase- or laccase-highly producing strain with an ability to strongly degrade lignin.
That is, the present invention is summarized as follows.
(1) A Coriolus hirsutus host cell transformed with a vector containing a basidiomycete-derived promoter region selected from the group consisting of ras and priA gene promoter regions from basidiomycetes.
(2) A host cell defined in (1) above, wherein the ras gene promoter region is derived from Coriolus hirsutus or Lentinus edodes.
(3) A Coriolus hirsutus host cell defined in (1) above, wherein the priA gene promoter region is derived from Lentinus edodes.
(4) A Coriolus hirsutus host cell defined in (1) above, wherein the vector further comprises a gene encoding a useful polypeptide, the gene being transcribably ligated to a site downstream of the foregoing promoter region.
(5) A Coriolus hirsutus host cell defined in above (3), wherein the said gene encoding a useful polypeptide is a gene coding for a lignin degrading enzyme such as manganese peroxidase, lignin peroxidase, or laccase.
(6) A process for producing a useful polypeptide comprising culturing the Coriolus hirsutus host cell recited in (1) above in a medium and recovering the formed useful polypeptide.
(7) A process defined in (6) above, wherein the useful polypeptide is a lignin degrading enzyme such as manganese peroxidase, lignin peroxidase, or laccase.
(8) An isolated DNA fragment containing a Coriolus hirsutus-derived ras gene promoter region.
(9) An isolated DNA fragment defined in (8) above, wherein the DNA fragment has a nucleotide sequence shown in SEQ ID NO:1 or a sequence that hybridizes to a sequence complementary to the nucleotide sequence under stringent conditions and has a promoter activity.
(10) A recombinant DNA, which contains a gene encoding a useful polypeptide and the DNA fragment recited in (8) above, the said gene being transcribably linked to the DNA fragment.
(11) A recombinant DNA defined in (10) above, wherein the gene encoding a useful polypeptide is a gene coding for a lignin degrading enzyme such as manganese peroxidase, lignin per-oxidase, or laccase.
(12) A DNA containing a Coriolus hirsutus-derived ras gene promoter sequence and ras gene sequence and has a nucleotide sequence shown in SEQ ID NO:2.
(13) A vector containing the DNA fragment recited in (8) above or the recombinant DNA in (10) above.
(14) A host cell transformed with the vector recited in (13) above.
(15) A host cell defined in (14) above, wherein the host is a basidiomycete.
(16) A host cell defined in (15) above, wherein the basidiomycete is Coriolus hirsutus
(17) A process for producing a useful polypeptide, comprising culturing the host cell recited in (13) above in a medium and recovering the formed useful polypeptide.
(18) A process defined in (17) above, wherein the useful polypeptide is a lignin degrading enzyme such as manganese peroxidase, lignin peroxidase, or laccase.